Application of indocyanine green–human serum albumin complex in fluorescence image-guided laparoscopic anatomical liver resection: study protocol for a randomized controlled trial

Abstract

Background

Indocyanine green (ICG) is a near-infrared fluorescent dye widely used for intraoperative navigation during liver surgeries because of its non-radioactive nature, high safety, and minimal impact on liver function. However, variability in its dosage and concentration and its low imaging success rates have limited its widespread application. To address these issues, we developed a novel ICG–human serum albumin (ICG-HSA) complex to enhance fluorescence visualization during laparoscopic anatomical liver resection.

Methods

This prospective, double-blind, single-center, randomized controlled trial will compare the fluorescence navigation effects of the novel ICG-HSA complex with the guideline-recommended ICG administration scheme. The study will involve patients aged 18 to 75 years with malignant liver tumors. The participants will undergo evaluations at specified time points, and data will be collected using an internet-based electronic data capture system. The primary outcome will be the effectiveness of intraoperative fluorescence imaging, assessed by three independent experts. The secondary outcomes will be conversion to open surgery, the total operative time, intraoperative blood loss, and long-term survival rates.

Discussion

The aim of using this novel ICG-HSA complex will be to improve the success rate of fluorescence navigation in liver resection by ensuring better stability and a longer liver retention time compared with free ICG. This study seeks to validate the clinical value of ICG-HSA in enhancing surgical precision and outcomes, ultimately promoting its broader clinical application. The results are expected to provide high-level evidence supporting the safety and efficacy of this new fluorescence imaging agent.

Trial registration

ClinicalTrial.gov NCT06219096. Registered on 1 December 2024.

Introduction

Background

Indocyanine green (ICG) is a contrast agent with near-infrared spectral properties [1]. Because of its non-radioactive nature, high safety, and minimal impact on liver function, ICG is widely used in the assessment of liver reserve function, diagnosis of liver diseases, and intraoperative navigation during liver surgeries [2]. In the field of anatomical liver resection, where ICG is most widely applied, it primarily achieves fluorescence visualization of liver segments or lobes via “positive staining” or “negative staining” techniques through the portal vein [3]. Compared with traditional techniques, ICG fluorescence staining not only delineates the boundaries of liver segments or lobes on the liver surface but also clearly shows the inter-segmental planes in the deeper parenchyma, especially in areas without hepatic veins. It has thus become an important technical means for precise anatomical liver resection [4–7].

Although laparoscopic anatomical liver resection (LALR) using near-infrared fluorescence (NIF) technology is widely used in the treatment of malignant liver tumors, there is still a lack of high-level clinical evidence to clarify the optimal timing and dosage of ICG injection. In 2021, the guidelines for fluorescence imaging technology in hepatobiliary surgery, led by Wang et al. [3], recommended “slow intravenous injection of 2.5 mg ICG via peripheral veins” when using ICG for negative staining. In 2023, Wakabayashi et al. [8] conducted a systematic review specifically addressing the timing and dosage of ICG for intraoperative navigation in liver surgery. After analyzing 26 studies on negative staining fluorescence imaging in liver resection, they found that the dosage of ICG varied significantly from 0.025 mg to 25 mg. It was discussed that the guideline-recommended dosage by Wang et al. [3] is not necessarily the “optimal” but rather the “most commonly used (without validation by research)” dose, as it is based on data from previous studies. In 2020, Xu et al. [9] reported that the success rate of negative staining in laparoscopic liver resection using 2.5 mg as the ICG injection dose was only 52% (14/27). Current research indicates [10, 11] that after intravenous injection, ICG rapidly binds to plasma proteins and is specifically taken up by hepatocytes, thus enabling real-time visualization of liver tumors and parenchyma. However, ICG also has some limitations, such as poor water solubility, susceptibility to concentration-dependent aggregation, and a short liver retention time, which restrict its widespread application to some extent.

To overcome the aforementioned limitations, we recently developed a novel complex formed by human serum albumin (HSA) and ICG (ICG-HSA complex). HSA is an endogenous protein naturally present in the human body. Studies have shown that HSA in aqueous solution can significantly improve the dispersibility and fluorescence performance of ICG [12, 13]. HSA can bind with ICG to form nanoparticles of approximately 4 to 7 nm in size, thereby reducing the number of free ICG monomers and preventing background interference caused by their free diffusion [14]. Compared with free ICG, ICG-HSA exhibits greater stability and a longer liver retention time. Based on these research advancements, our center has explored a novel application scheme of NIF negative staining using the ICG-HSA administration method in LALR. We pre-conjugated ICG molecules with albumin in vitro and injected them into the body in the form of a stable conjugated fluorescent molecular complex to mitigate the effects of free ICG molecules.

Objective

The primary objective of this study is to evaluate the effectiveness of the novel ICG-HSA complex in enhancing fluorescence visualization during laparoscopic anatomical liver resection compared to the guideline-recommended ICG administration scheme.

Specific objectives include the following: (1) To compare the effectiveness of intraoperative fluorescence imaging between the ICG-HSA complex and the standard ICG method.

To assess the safety of the ICG-HSA complex in patients undergoing laparoscopic anatomical liver resection. (2) To evaluate secondary outcomes including conversion to open surgery, total operative time, intraoperative blood loss, and long-term survival rates. (3) To determine the stability and liver retention time of the ICG-HSA complex compared to free ICG.

Hypotheses: (1) The ICG-HSA complex will provide superior fluorescence visualization compared to the standard ICG method. (2) The ICG-HSA complex will be safe for use in patients undergoing laparoscopic anatomical liver resection. (3) The use of the ICG-HSA complex will result in improved surgical outcomes and long-term survival rates compared to the standard ICG method.

Trial design

This study is a prospective, double-blind, single-center, randomized controlled trial with a parallel group design. Patients will be randomized in a 1:1 allocation ratio to either the experimental group receiving the novel ICG-HSA complex or the control group receiving the guideline-recommended ICG administration. The framework of this trial is to compare the efficacy and safety of the novel ICG-HSA complex against the standard ICG administration method in fluorescence-guided laparoscopic anatomical liver resection. The primary aim is to demonstrate superiority in terms of the effectiveness of intraoperative fluorescence imaging.

Methods

Study design

This prospective, double-blind, single-center, randomized controlled trial will be conducted at the Liver Transplantation Center of West China Hospital, Sichuan University. The aim of this study is to compare the fluorescence navigation effects of our center’s novel ICG-HSA administration scheme with the current guideline-recommended scheme for NIF-negative staining in LALR [3] as well as to analyze the safety and efficacy of this new administration scheme.

Study population

Inclusion criteria

Patients must meet all of the following criteria to be eligible for inclusion in this study: (1) age of 18 to 75 years with a malignant liver tumor requiring LALR, (2) preoperative Child–Pugh class A or B liver disease, (3) no contraindication to laparoscopic liver resection, (4) expected survival of ≥ 3 months, (5) Eastern Cooperative Oncology Group performance status score of 0 or 1, and (6) laboratory testing within 7 days prior to enrollment meeting the following conditions: white blood cell count of ≥ 2.5 × 10⁹/L, absolute neutrophil count of ≥ 1.5 × 10⁹/L, platelet count of ≥ 75 × 10⁹/L, hemoglobin level of ≥ 90 g/L, international normalized ratio of ≤ 1.5 × upper limit of normal (ULN), serum creatinine level of ≤ 1.5 × ULN, and total bilirubin level of ≤ 1.5 × ULN.

Exclusion criteria

Patients who meet any of the following criteria will be excluded from participation in this study: (1) no obvious ischemic demarcation line after intraoperative blocking or isolation of the target liver pedicle, or liver fluorescence reaching an intensity that interferes with the operation before intraoperative intravenous injection of ICG; (2) ICG retention rate at 15 min of ≥ 20%; (3) severe cardiopulmonary disease precluding surgery under general anesthesia; (4) clinically significant ascites or pleural effusion; (5) active bleeding or coagulation dysfunction; (6) hepatic encephalopathy; (7) allergy to ICG; (8) history of gastrointestinal bleeding within the past 6 months or a tendency for gastrointestinal bleeding; (9) severe gastroesophageal varices requiring treatment; (10) objective evidence of past or present severely impaired lung function; and (11) any clinical or laboratory abnormalities that may affect the safety evaluation.

Study drugs

For this study, we have developed a novel ICG-HSA complex for use in fluorescence image-guided LALR. The clinical formulation of the ICG-HSA complex was designed based on the results of in vitro validation experiments. The complex was prepared using commercially available HSA (10 g/50 mL) and ICG (25 mg/10 mL). Given that the molar mass of HSA is approximately 66,500 g/mol [15] and the molar mass of ICG is 774.96 g/mol [10], we calculated the required volumes of each component to achieve a molar ratio of ICG:HSA = 1:6. This ratio was determined to be optimal for clinical use. To prepare the clinical formulation, 2 mL of the commercial HSA injection and 0.3 mL of the commercial ICG solution were mixed together. The mixture was then diluted with 15 mL of normal saline to maintain the desired molar ratio while providing a sufficient volume for clinical administration. The final ICG-HSA complex solution was stored in a 20-mL syringe (Fig. 1), which was connected to an intravenous infusion pump to ensure uniform injection during the surgical procedure.

Fig. 1.

Configuration diagram of ICG-HSA complex. A The raw materials used to create ICG-HSA are readily available: HSA injection (10 g/50 mL) and ICG injection (25 mg/10 m). The molar ratio of ICG:HSA = 1:6; therefore, the required volume of the finished HSA injection is approximately 0.02 mL, and the finished ICG injection is approximately 0.031 mL. B HSA injection (2 mL) is thoroughly mixed with ICG injection (0.3 mL) at room temperature. C The ICG-HSA complexes are diluted with 15 mL of normal saline to form the final study drug

Randomization

This study will employ a customized electronic network central randomization system (https://study.empoweredc.com/) for block randomization allocation using a block length of 4. Patients will be randomly assigned in a 1:1 ratio to either the treatment group (study medication group) or the control group (guideline medication group). After enrolling patients who meet the inclusion criteria, the surgical team will complete the target hepatic pedicle occlusion operation. A dedicated research nurse will then perform the registration and randomization in the randomization system according to the order of trial entry (note: this must be performed according to the order of enrollment because the first to enroll may not be the first to undergo surgery). The system will automatically assign the corresponding random number and group to the patient.

Blinding

This study will adopt a double-blind design for both the surgical team (outcome assessors) and the patients. The random group assignments will be visible only to the dedicated research nurse, who will provide the different ICG administration schemes for the two groups based on the random numbers and group assignments during the surgery. An opaque curtain will be used to cover the anesthesia operation area at the patient’s head side, thereby blinding the surgical operators to the random group assignments. The entire process of grouping and administration will be visible only to the dedicated research nurse (Fig. 2).

Fig. 2.

Diagram of the blinding method. A An opaque curtain is used to cover the anesthesia operation area at the patient’s head side, preventing the surgical team from knowing the group assignment. The entire process of grouping and administration is visible only to the dedicated randomization nurse. B The nurse uses an infusion pump to administer the ICG-HSA complex solution through the patient’s central venous catheter

Concealment of group assignments

To reduce assessment bias, the outcome assessors and data collectors will be blinded such that they are unaware of the patients’ group assignments, thereby minimizing the impact of bias on the results. The primary endpoint will be determined by three independent experts in the field of fluorescence-guided laparoscopic liver resection, each of whom will independently evaluate and score the fluorescence imaging results in the surgical video recordings.

Unblinding principles

Emergency unblinding may only be performed if the patient develops an adverse event or if the investigator deems it necessary to clarify the patient’s specific treatment group for further management of the situation. The investigator can log into the randomization system and unblind the patient by entering the password. The unblinding page must document the reason for emergency unblinding, the time, and the personnel involved in the unblinding process.

Criteria for terminating the study

1. Serious adverse event: If a patient develops a serious adverse event during the study that significantly impacts their health and may be related to the study intervention, it may be necessary to consider terminating the study.

2. Lack of efficacy: If, after a certain period of observation and treatment, the study intervention has failed to achieve the expected efficacy or no significant differences have been observed compared with the control group, it may be necessary to consider terminating the study.

3. Exceeding predetermined safety limits: If the frequency or severity of adverse events caused by the study intervention exceeds the predetermined safety limits, the study may need to be terminated to protect the safety of the patients.

4. Poor compliance: If patients are unable to follow the study protocol requirements or lack necessary cooperation or compliance during the study, and this significantly impacts the reliability of the data, it may be necessary to consider terminating the study.

The criteria for terminating the study should be established under the guidance of the study protocol and the ethics review board, and these criteria should be clearly documented in the study protocol in advance. Upon terminating the study, relevant institutions and participants should be promptly notified, and necessary data analysis and interpretation should be conducted.

Principles for handling dropouts

1. Recording and statistics: For each patient who drops out, the reason for dropout will be accurately and promptly recorded, and a statistical analysis will be conducted. This will help to understand the overall situation of dropouts as well as the proportions and trends of different reasons for dropping out.

2. Adverse events: For dropouts caused by serious adverse events, the severity and relevance of the event will be carefully evaluated, and consideration will be given to whether the trial needs to be halted or the protocol adjusted. This will require comprehensive consideration of patient safety and the scientific validity of the trial.

3. Voluntary withdrawal: For patients who voluntarily withdraw from the trial, consultations will be conducted as early as possible to understand their reasons for withdrawal, and these reasons will be recorded. This will help to analyze the patients’ acceptance of the trial intervention and identify potential areas for improvement.

4. Exclusion criteria: Patients who meet the exclusion criteria will be screened and excluded before the trial begins. This will prevent patients who do not meet the trial requirements from interfering with the trial results.

Trial interventions

Based on preoperative three-dimensional reconstruction imaging, an anatomical liver resection plan will be formulated to resect the liver segment or lobe containing the tumor. All surgeries will be performed by the same hepatobiliary surgical team. The surgeons that will be involved in this study have more than 10 years of experience and have each completed more than 100 laparoscopic hepatectomies. After induction of general endotracheal anesthesia, the patients will be placed in the supine position for laparoscopic hepatectomy. The intra-abdominal pressure will be set at 10 to 14 mmHg. Central venous pressure will be monitored and maintained below 5 cmH₂O. An ultrasonic scalpel (Harmonic Scalpel; Ethicon, Cincinnati, OH, USA) will be used to adequately mobilize the hepatic ligaments. We will use the guideline-recommended “negative staining method” to complete the LALR [3]. The hepatic pedicle of the lobe or segment planned for resection will be isolated and occluded using the Glissonean pedicle transection technique after confirming the diseased area.

After completing the above steps, patients assigned to the control group will receive a peripheral intravenous injection of 1 mL ICG solution (2.5 mg/mL) administered by a dedicated nurse, followed by observation of liver fluorescence imaging. Patients assigned to the trial group will receive a steady peripheral intravenous infusion of the prepared novel ICG-HSA complex at a rate of 1 mL/min using an intravenous infusion pump, administered by a dedicated nurse. The infusion will be stopped when fluorescence imaging appears on the liver surface under the fluorescence laparoscopic system (FloNavi® 214 K; OptoMedic Technologies, Foshan, Guangdong, China) (Fig. 3). Intraoperative ultrasound will be used to confirm whether the tumor is located in the liver segment of fluorescence imaging.

Fig. 3.

Schematic of a fluorescence “negative staining” laparoscopic anatomical left hemihepatectomy

Once it has been confirmed that the tumor is located in the non-fluorescent area and that the resection margin is sufficient, the liver segment will be resected along the fluorescence boundary under full liver blood flow occlusion using the Pringle maneuver. After completing the liver resection, the liver specimen will be extracted through the umbilical incision. The pneumoperitoneum will then be deflated, puncture sites sutured, and operation completed.

Primary endpoint

The primary outcome of this trial will be the effectiveness of intraoperative fluorescence imaging. After the patient is discharged, the research team will provide the complete unedited surgical video to three experts in the field of fluorescence-guided laparoscopic liver resection. These experts will score the video according to our established scoring criteria (Fig. 4), and the average score from the three experts will be used as the primary outcome for the patient.

Fig. 4.

Expert scoring criteria for fluorescence imaging effectiveness. Based on a maximum score of 6 points, the experts will use the complete unedited surgical video to make corresponding deductions or additions according to the scoring standards shown in the figure

Secondary endpoints

The secondary endpoints will be conversion to open surgery, total operative time, intraoperative blood loss, intraoperative transfusion, tumor resection margins, post-hepatectomy liver failure [16], length of postoperative hospital stay, incidence of complications (postoperative complications will be graded based on severity according to the Clavien–Dindo classification [17]), unplanned reoperation rate, postoperative mortality rate, overall survival time, and disease-free survival time.

Data collection and follow-up

Data collection will be performed prospectively for all patients, encompassing their medical history, physical examination findings, laboratory results, pathological assessments, perioperative clinical details, and any complications. Information will be recorded on paper datasheets and stored securely. The patients will undergo evaluations at the time of inclusion, at 1 week postoperatively, and at 3, 6, 12, 24, and 36 months postoperatively. Follow-up by telephone or outpatient visits will occur at 1 month postoperatively and then at 3, 6, 12, 24, and 36 months postoperatively. Throughout follow-up, liver function tests and abdominal ultrasound examinations will be conducted for a duration of up to 3 years or until the patient’s death. The schedule of enrolment, interventions, and assessments for this trial is summarized in the SPIRIT figure below (Fig. 5). This figure provides an overview of the overall timeline and key activities at each visit, from initial eligibility screening through to study close-out.

Fig. 5.

SPIRIT schedule of enrolment, interventions, and assessments. − t1: screening period (14 days before surgery); 0: surgery day; t1: 1 week postoperatively; t2: 1 month postoperatively; t3: 3 months postoperatively; t4: 6 months postoperatively; t5: 12 months postoperatively; t6: 24 months postoperatively; t7: 36 months postoperatively;¹Pulse, blood pressure, body temperature. ²Routine blood tests (WBC, RBC, HGB, PLT, NL, LY) and urine routine tests (LEU, BLD, GLU, PRO). ³Blood biochemistry (ALT, AST, GGT, TBIL, ALP, Urea, Cr, GLU). ⁴Only for women of childbearing age. ⁵ICG administration protocol (experimental group, control group), conversion to open surgery, liver parenchyma disconnection time, total operation time, intraoperative bleeding volume, specimen cutting margin (malignant tumor). ⁶Complications (incidence, Clavien–Dindo classification [17], treatment measures), postoperative mortality, postoperative liver function recovery

The measurement time points and methods for various short-term and long-term secondary endpoints are detailed in Table 1. All clinical data, including patient demographics, surgical details, tumor characteristics, liver tumor clinical staging, perioperative observation indicators, and results from short-term and long-term follow-up assessments, will be entered and collected using an internet-based electronic data capture (EDC) system (https://study.empoweredc.com/) and centralized at the principal investigator’s center. The principal investigator and co-investigators, with assistance from the research assistants, will conduct continuous clinical data monitoring as well as interim and final analyses.

Table 1.

Measurement times and methods of short- and long-term secondary indicators

	Indicator name	Timing of measurement	Method of measurement
Short-term secondary indicators	Surgical time	At the end of the surgery	Based on the recorded duration of the surgery
	Surgical blood loss	At the end of the surgery	Based on the recorded blood loss during surgery
	Laparotomy conversion	At the end of the surgery	Based on the intraoperative situation, determine whether to convert to open surgery
	Time of hepatic parenchyma transection	After the surgery	Organize a designated person to review the surgical video and record its duration afterwards
	The number of hemostatic clamps deployed on the sectioned surface of the liver	At the end of the surgery	Organize a dedicated person to review the surgical video and record the findings afterwards
	Resection margin of tumor	At the end of the surgery	Organize a designated person to use a caliper to measure the resection margin of the specimen
	Post-surgical complication condition	After the surgery and returning to the room	Organize a dedicated person to document post-surgical complication status
	Post-surgical liver function status	After the surgery and returning to the room	Regularly measure postoperative liver function, coagulation, and other biochemical indicators
	Postoperative recovery time of intestinal function	After the surgery and returning to the room	Organize a dedicated person to record the time of postoperative return of bowel function
	Postoperative hospital stay	At the time of discharge	Organize a dedicated person to record the postoperative hospital stay duration
Long-term secondary indicators	Overall survival time	After discharge (once every 3 months)	Using the specialized disease EDC (Electronic Data Capture) data management software's follow-up center function to conduct WeChat, questionnaire, and phone follow-up records
	Disease-free survival	After discharge (once every 3 months)

Statistical analyses

Sample size calculation

The sample size has been calculated based on the primary outcome of intraoperative fluorescence imaging effect scores. Assuming a mean score of 4 in the control group and an expected mean score of 4.5 in the experimental group, and with a common standard deviation of 0.8, a sample size of 64 patients per group would provide 80% power to detect a statistically significant difference between groups using a two-sided t-test at a significance level of 0.05 (NCSS and PASS 15 statistical software; NCSS, LLC, Kaysville, UT, USA). Allowing for a 10% dropout rate, 142 patients (71 per group) will be recruited.

Statistical methods

Statistical analysis will be performed using SPSS 26.0 software (IBM Corp., Armonk, NY, USA). Continuous variables will be expressed as mean ± standard deviation or median (interquartile range) and compared using the independent-samples t-test or Mann–Whitney U test. Categorical variables will be expressed as number (percentage) and compared using the chi-square test or Fisher’s exact test. The primary outcome of intraoperative fluorescence imaging scores will be compared between groups using the independent-sample t-test. Secondary short-term outcomes such as conversion to open surgery, total operation time, intraoperative blood loss, tumor margins, postoperative liver failure, length of hospital stay, complication rate, unplanned reoperation rate, and mortality will be compared between groups using appropriate statistical tests based on the type and distribution of the data. Long-term outcomes of overall survival and disease-free survival will be analyzed using the Kaplan–Meier method, and differences between groups will be assessed using the log-rank test. Multivariable Cox proportional hazards models will be used to identify independent prognostic factors associated with overall and disease-free survival. All statistical tests will be two-sided, and p-values of < 0.05 will be considered statistically significant. Intention-to-treat analysis will be used, with all randomized patients included in the analysis according to their assigned group. Per-protocol analysis will also be performed as a sensitivity analysis. Subgroup analyses based on factors such as the type of liver resection may be conducted if deemed appropriate.

An interim analysis will be performed to assess safety and efficacy when 50% of the planned sample size has been enrolled. The trial may be stopped early if there are significant differences in safety outcomes between groups or if the efficacy boundary is crossed. The final analysis will take place once complete survival data have been collected after the 3-year follow-up of the last enrolled patient.

Quality control of study data

The principal investigator and co-investigators, with the assistance of the research assistants, will conduct continuous clinical data monitoring as well as interim and final analyses using the EDC system (https://study.empoweredc.com/). The researchers will complete and submit the online version of the case report form promptly after each patient’s visit. The researchers must respond to queries from monitors, data managers, and medical reviewers in a timely manner. After data cleaning has been completed, the researchers will sign off to confirm the data for each patient.

Data review and database lock

Upon completion of the clinical trial, the study lead, statisticians, and data managers will jointly conduct a pre-statistical analysis data review. The key aspects will be determining the analysis dataset for each case (including the full analysis set, per-protocol set, and safety set), assessing missing values, and handling outliers. Once the data review confirms that the database is accurate, the database will be locked. After locking, the database cannot be modified arbitrarily, and any modification decisions must be documented. The locked database will be properly preserved for future reference and handed over to statisticians for analysis.

Data safety monitoring

A data safety monitoring plan will be developed based on the level of risk. All adverse events will be recorded in detail, appropriately managed, and tracked until resolved or stabilized. Serious adverse events and unexpected events will be reported promptly to the ethics review board, health authorities, and drug regulatory agencies as required. The principal investigator will periodically conduct cumulative reviews of all adverse events and, if necessary, convene investigator meetings to assess the risk and benefits of the study. In double-blind trials, emergency unblinding may be performed if necessary to ensure the safety and rights of the patients.

Ethics and dissemination

The study will be conducted in accordance with the ethical principles set out in the Declaration of Helsinki and will be consistent with the International Council for Harmonisation/Good Clinical Practice as well as the regulatory requirements for participant data protection. It will be conducted in West China Hospital, which has been approved by the Ethics Committee of West China Hospital, Sichuan University. All participants will receive the information necessary to provide informed consent, including key details about the clinical trial such as financial costs, the operative procedure, benefits, and risks. They will be entitled to withdraw from the study at any time without providing a reason. We will share the trial results with the public within 1 year after completion of the clinical trial and publish the results in a peer-reviewed journal. However, systematic individual patient data sharing will not be performed.

Discussion

ICG is a typical cyanine NIF dye that has long been approved for clinical use by the FDA. Under normal conditions, ICG is non-fluorescent. However, when ICG is excited by near-infrared light, it emits fluorescence, making it useful for fluorescence imaging [1]. Because of its liver-targeted NIF properties, ICG has recently been utilized for navigation in precise liver resection, becoming the only method currently capable of achieving precise liver resection as recommended by various guidelines and expert consensus [3, 8]. The metabolism of ICG in the body mainly occurs in three phases: the intravascular phase, the hepatic metabolism phase, and the excretion phase [18].

In the intravascular phase, ICG binds to plasma macromolecules, with most of it being confined within the blood vessels until it is absorbed by the liver and excreted into the bile. Most ICG binds to plasma proteins, particularly albumin and α- and β-lipoproteins, and its lipophilic components interact with the hydrophobic regions of these proteins. This interaction does not alter the protein structure but creates a non-toxic interface and reduces dye leakage. The tendency of plasma macromolecules to aggregate with ICG in the blood is a dynamic process that involves competition and equilibrium. Bound ICG is confined and retained within the blood vessels, continuously circulating and being delivered to the liver, where it is specifically taken up by the liver [19].

In the hepatic metabolism phase, ICG is transported to the hepatic sinusoids by plasma albumin and then into hepatocytes via organic anion-transporting polypeptide 1B3 and sodium taurocholate co-transporting polypeptide on the hepatocyte surface. It is then transported into the bile by glutathione S-transferase within the cytoplasm. The clearance rate of ICG in the liver is rapid, ranging from 18 to 24% per minute, which is a result of the compound being confined within the intravascular space and the breakdown products of ICG not being metabolites [19, 20]. This dye is exponentially cleared from the body within the first 10 to 20 min after administration. The half-life typically ranges from 3 to 4 min depending on the vascularization of the target organ. This rapid clearance rate allows for multiple injections of the dye during surgery. The blood concentration of bromsulphalein, a clinical dye also used in early liver blood flow concentration studies, at 45 min is equivalent to that of ICG at 20 min.

At high doses, the clearance of ICG follows standard Michaelis–Menten enzyme kinetics. This close correlation with standard enzyme kinetics suggests a high specificity of hepatocyte carriers for ICG. The relative affinity of hepatocytes for ICG further facilitates the rapid clearance of this substance from the body and implies that similarly structured compounds, or analogs, might be processed similarly. Human serum albumin’s role in the metabolism of small molecule drugs is characterized by its three-dimensional structure, which contains both hydrophobic and hydrophilic domains, along with a rich presence of charged amino acids. This allows albumin to carry various drugs with different physicochemical properties without the need for additional compounds.

Issues have been identified in the use of ICG for hepatic parenchymal transection imaging. Despite the high affinity of ICG for plasma proteins, Mordon et al. [21] proposed that some ICG might bind to the endothelium because of its affinity for phospholipid bilayers. This has been demonstrated in vitro, where changes in the microenvironment can make ICG more hydrophilic than plasma proteins, potentially increasing its binding tendency when used in the vascular endothelium. To validate the safety of the ICG-HSA complex, we conducted a prior case series study [22], which confirmed its clinical safety with no significant adverse events reported. Since the complex is formed by pre-binding ICG and HSA in vitro without altering their biological or physicochemical properties, the risk of introducing new or unexpected side effects is minimal. For this trial, we have established a comprehensive adverse event monitoring protocol, including measures to detect hypersensitivity reactions or other side effects and procedures for emergency unblinding and patient management.

In summary, by pre-binding ICG molecules to HSA to form a stable conjugate complex that remains confined within the vasculature and is then selectively taken up by the liver, we aim to prevent ICG from binding to other macromolecules or existing in a free state, which can cause metachromasia. This approach reduces the likelihood of ICG molecules being degraded by free radicals such as free oxygen and sulfur, which can destabilize the double-bond structure, leading to loss of function and impaired fluorescence performance. Therefore, we have designed and implemented the ICG-HSA complex and registered to conduct the herein-described prospective randomized controlled study.

ICG primarily relies on positive or negative staining techniques via the portal vein route to achieve fluorescence visualization and navigation for liver segments or lobes [4]. Successful ICG fluorescence staining can delineate the boundaries of liver segments or lobes not only on the liver surface but also deep within the liver parenchyma, particularly in areas without hepatic veins. The positive staining method involves directly puncturing the target portal vein and injecting an appropriate amount of ICG to visualize the corresponding portal vein territory. This requires high skills in intraoperative ultrasound-guided puncture, which can be challenging for beginners. Conversely, the negative staining method involves blocking the target hepatic pedicle and then injecting ICG via a peripheral vein. The negative staining method has a shorter learning curve because of the use of traditional Glisson’s sheath dissection techniques combined with peripheral intravenous administration [23]. For greater clinical applicability and higher imaging success rates, we chose to use the negative staining method exclusively in this study.

Research to date has indicated that compared with traditional white-light laparoscopic liver resection, ICG fluorescence-guided precise liver resection ensures safer surgical margins and improves the R0 resection rate of tumors. It also offers advantages such as shorter operation times, reduced intraoperative blood loss, lower postoperative complication rates, and improved survival [6, 7, 24]. However, because of the low imaging success rate of existing fluorescent agents and the significant variability in the dosage and concentration of ICG reported by different centers [8, 9], the widespread application of ICG fluorescence-guided technology in real-world settings has been suboptimal. We plan to conduct exploratory subgroup analyses in this study to stratify results based on tumor type, stage, and patient characteristics (e.g., age, liver function, and tumor location), to identify specific patient populations that may benefit most from the use of the ICG-HSA complex. This will provide hypotheses for future research. The primary objective of our study is to develop a new fluorescent imaging agent that is safe for human use, that can achieve a fluorescence navigation success rate of > 90% during liver resection, and whose imaging effectiveness is not affected by liver texture. We aim to achieve this through the combination of basic chemical research and clinical studies. Additionally, this randomized controlled trial will validate the clinical value of the new ICG-HSA fluorescence navigation technique in precise curative resection of liver cancer, ultimately promoting its widespread clinical application.

Trial status

The trial started recruitment in December 2023. The trial is expected to be completed by December 2028. Protocol Version: Version 9.1, 2023–11–21.

Abbreviations

ICG

Indocyanine green

LALR

Laparoscopic anatomical liver resection

HAS

Human serum albumin

EDC

Electronic data capture

NIF

Near-infrared fluorescence

FDA

Food and Drug Administration

Authors’ contributions

Qingyun Xie: performed surgeries, conceptualization, methodology, resources, writing—original draft, writing—review and editing. Fengwei Gao: performed surgeries, conceptualization, methodology, resources, writing—original draft, writing—review and editing. Xiaoyun Ran: stability testing of reagents, preparation of reagent protocols. Xin Zhao: literature search, patient management, data entry. Manyu Yang: literature search, patient management, data entry. Kangyi Jiang: investigation, supervision, writing. Tianyang Mao: literature search, patient management, data entry. Jiayin Yang: investigation, supervision, writing. Kun Li: stability testing of reagents, preparation of reagent protocols. Hong Wu: investigation, supervision, writing. All authors have given final approval for the version to be published.

Funding

This work was supported by grants from the Horizontal Science and Technology Project of West China Hospital, Sichuan University (Grant No. HX-H2402041).

Declarations

Ethics approval and consent to participate

This prospective study was approved by the Medical Ethics Committee on Biomedical Research, West China Hospital of Sichuan University (Ethics number: 2023 review (2121)). All methods were carried out in accordance with relevant guidelines and regulations. Research involving human participants, human material, or human data is conducted in accordance with the Declaration of Helsinki. The authors confirmed that informed consent was obtained from all subjects.

Consent for publication

Not applicable.

Competing interests

All of the results will be published. There are no financial interests in this trial and none of the parties concerned has right of veto.